Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929–939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH2 and -CO2H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH2 (GelKNH2) and -CO2H (GelKCOOH) halves were overexpressed in Escherichia coli. GelK and GelKNH2 were present in both the soluble and membrane fractions of E. coli, whereas GelKCOOH was only present in the soluble fraction. GelK catalysed the transfer of [14C]glucuronic acid from UDP-[14C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by β-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PPi-lipid moiety is needed for enzymic activity. Recombinant GelKNH2 and GelKCOOH did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.